The overall objective of this multi-project HIVRAD program application is to elicit neutralizing antibodies that target the CD4 binding site (CD4bs) of primary HIV-1 envelope (Env) glycoproteins. This P01 program proposal consists of three major projects, plus two cores to support the activities of major projects. The following is a summary of major activities as proposed in different Projects/Cores of this program. Goal 1: To organize and manage a highly interactive and productive research team (Core B). Goal 2: To understand how the variation in HIV-1 R5 Envs affect tropism, neutralization and vaccine development (Project 1). HIV-1 R5 envelopes vary extensively in their capacity to infect macrophages. We propose to investigate the impact of variation in macrophage tropism (mac-tropism) on other biological properties associated with Env including neutralization sensitivity. Goal 3: To study how the variation of antigenicity of CD4bs will affect the neutralization sensitivity and immunogenicity of primary Env proteins (Project 2). The CD4bs antigenicity of several panels of primary Envs, each with their own unique biological features, will be probed by mAbs. We will examine whether high CD4bs antigenicity and high sensitivity to CD4bs mAb mediated neutralization will lead to high immunogenicity for key representative Env using the DMA prime-protein boost immunization approach. Goal 4: To study the modification of receptor binding site as an approach to HIV-1 vaccine design (Project 3). We will test whether changes resulting from specific glycan modifications will lead to increased stability or accessibility of conserved epitopes in the receptor binding site and whether greater accessibility of these conserved sites will enhance their function as immunogen to elicit cross-reactive NAb responses. Goal 5: To provide support to major projects on structure analysis of Env and to study the structure of novel antigens, and antigen-antibody interactions (Core A). PROJECT 1: Variation in HIV-1 R5 Envs: Consequences for tropism, neutralization and vaccines (Chapham, P) PROJECT 1 DESCRIPTION (provided by applicant): HIV-1 R5 viruses vary considerably in phenotypes, including macrophage-tropism and sensitivity to neutralizing antibodies. Thus, highly mac-tropic envelopes (envs) exploit low amounts of CD4 and/or CCR5 for infection and contrast with non-mac-tropic R5 envs that require high CD4 levels and infect macrophages inefficiently. The capacity of highly mac-tropic envs to use low CD4/CCR5 suggests that such variants may confer a broader tropism among other CD4+ cell types that express low receptor levels and may have an advantage during virus transmission. Our recent data, using neutralizing monoclonal antibodies (mAbs) and entry inhibitors, strongly suggests that highly mac-tropic R5 envs carry a more exposed CD4 binding site (CD4bs) and may be vulnerable to neutralizing antibodies (NAbs) induced by vaccines. We propose to extensively investigate tropism and neutralization sensitivity of diverse R5 envs in the following four aims: Aim 1. The effect of HIV-1 R5 macrophage-tropism on infection of different CD4+ cell populations. We will examine whether highly mac-tropic R5 envelopes confer a broader tropism among CD4+ T-cell populations. Aim 2. Further analysis of the envelope determinants for macrophage infection and use of low CD4 in clade B and non-clade B envelopes. We will analyze clade B and non-clade B envs to identify determinants of tropism and to further examine the role of the CD4 binding loop. Aim 3. Investigation of the impact of R5 env variation in macrophage-tropism on sensitivity to neutralizing antibodies. We will investigate how R5 env variation impacts on sensitivity to HIV-1 + human sera, neutralizing mabs and vaccine induced neutralizing antibodies. Aim 4. Development and characterization of bacterially-produced, unglycosylated protein constructs that mimic the CD4bs. We have designed and produced CD4bs mimics that carry the critical elements of the CD4bs in an unglycosylated peptide that can be produced in bacteria. These CD4bs mimics bind CD4 and b12. Their structure and capacity to induce neutralizing antibodies will be investigated. We expect to provide important insights into (1) the variation of R5 envs for tropism and neutralization sensitivity, (2) the env amino acids and structural determinants involved in varying tropism and neutralization sensitivity and (3) the impact of this variation in the design of novel env-based vaccines.